Early life stress (ELS) can disrupt the development of nervous, cardiovascular, metabolic, and immune systems, leading to poor physical and mental health throughout the lifespan. The rising prevalence of ELS in preschool children is a major public health concern, epidemiologically linked with higher rates of youth suicides, dating violence, drinking, and drug abuse. Individuals respond to acute stress by releasing cortisol; thus, hair cortisol concentrations (HCC) can quantify the exposures to recurrent or chronic stress in children. Our pilot data on HCC revealed various factors that are associated with chronic stress in preschool children. We propose hair oxytocin concentrations (HOC) as a summative measure of supportive and/or socially affiliative experiences. Specific aims of the proposed study are: (1) to validate novel assays for measuring HCC and HOC in 1200 preschool children and their parents, in order to quantify their cumulative exposures to stressful vs. supportive early experiences; and (2) to measure hair growth rates, thereby delineating the time periods related to such exposures and their hair composition (protein, lipid, water content) to interpret and/or adjust the HCC and HOC values obtained from the preschool children and their parents. We will collect hair samples from children ages 1-5 years (n=1200) and at least one accompanying parent (n=1200). From parents we will obtain their child's demographic, developmental, health and environmental data. Hair samples will be processed using our novel extraction methods and analyzed using specific enzyme- linked immunosorbent assays (ELISA) for HCC and HOC; these assays will be validated using tandem mass spectrometry (LC/MS-MS). We will measure hair growth rates using the standard phototrichogram technique and hair composition using direct spectrophotometric detection. These data will establish the reference norms for HCC and HOC in preschool children and their hair growth rates will identify the time periods that these HCC or HOC values represent. We will examine the natural variations in HCC, HOC, and hair growth rates in relation to age, sex, race, ethnicity, parental, socioeconomic, or other environmental factors. HCC and HOC values obtained from the same hair sample will also allow us to examine oxytocin-mediated regulation of the cortisol responses to ELS. We have a multidisciplinary team (clinician, psychologist, sociologist, biochemists, statistician), the equipment, the experience, and pilot data to successfully complete the proposed project. Completion of this research will provide age-specific reference norms for HCC, HOC, and hair growth rates in healthy preschool children. This project will address fundamental gaps in our current knowledge and establish a scientific framework to investigate long-term physical and mental health consequences of early childhood stress, following physical or psychological trauma, traumatic/toxic stress, violence-related injury, child maltreatment, and acute or critical illnesses ? identified as a high-priority research area by NICHD.